The importance of cancer research and the inherent complexity of its challenges make it very important to design strategic, innovative approaches. Investigating the accumulation of multiple abnormalities per cell at the molecular level has prognostic value in cancer research. We propose to develop the capability to study this issue with optimal quantitative ability, discrimination and throughput by (a) focusing our proprietary technologies towards the goal of this solicitation; (b) developing the next-generation imaging cytometry instrument, able to bypass previous imitations; (c) raising the much needed optics/hardware/software /biology/clinical application continuum to a new level. We will concentrate on the imaging of 5-10 molecular species simultaneously within the same cancer cell, with a new approach and instrument that we propose to develop, based on our previous advances in acousto-optic technologies and multispectral imaging. The aims are largely technologic, but their implementation will allow us to (a) elucidate critical sequences of genetic evolutionary changes in solid tumors that are responsible for increasing cancer aggressiveness; (b) identify the steps in the sequence that are most closely associated with cellular acquisition of the capacity to metastasize, and (c) develop a practical overall approach for the timely performance of relevant measurements on individual tumors, analysis of the data, and characterization of each tumor with respect to the degree of its advancement along its particular genetic evolutionary pathway, so that this information can be used for prognosis and adjuvant treatment planning. The ultimate clinical challenge is the elimination of false negatives and false positives in diagnostics, leading to individually optimized treatment and very significant savings. The technological task specifically addressed is the design and development of the ability to quantitative simultaneously, in the same cell, a sufficiently large number of molecular species to enable a qualitative leap in diagnosis and treatment. The emphasis is on fluorescence, due to its very high specificity for labeling intracellular features, and the central new technology is acousto-optic tunable filters, a tool whose use in microscopic imaging we perfected and patented. Specific aims include (1) the building of a prototype instrument with the desired new functionality; (2) the implementation of a second, more user-friendly workstation in our cancer research laboratories; (3) the thorough testing of the latter instrument in experiments focusing on molecular-level prognostic factors for tumor progression within single cells of individuals' tumors, and (4) the development of a productized customizable version of the instrument embodying the new technology, multispectral imaging, ready for use on an array of problems by other researchers. A collaborative, multidisciplinary team is proposed for implementation of these goals.